Radiation Detection in Herbals
12
Herbs and herbal products are in use as food
supplements because of their unique nutritional
ingredients. Collection of herbs for medicinal
purposes and long-term storage in dried form are
quite common practices. Over the last two decades,
irradiation of herbs and products with g-rays has
emerged as a leading sterilization method against
microbial deterioration or insect infestation during
storage and transport, to preserve hygienic quality,
ensure shelf life, as well as to reduce public health
threat. Unlike other quarantine treatments such as
thermal treatment and fumigation with environ-
mentally disruptive toxic chemicals pose an occu-
pational health hazard, but irradiation technique is
considerably rapid, cost-effective, and offers a
broader spectrum of sanitizing applications for dry
ingredients. In view of the increasing use of irra-
diation technology for preservation of herbs, the
availability of analytical techniques for identifying
irradiated or nonirradiated herbs is of much impor-
tance for regulatory authorities and consumers to
increase confidence in radiation processing tech-
nology. Several detection methods, broadly cate-
gorized as chemical, physical, and biological, have
been developed for the identification of irradiated
herbs and herbal products. Among the physical
techniques, pulsed photo-stimulated luminescence
(PPSL), thermoluminescence (TL), and electron
paramagnetic spin resonance (EPR/ESR), spec-
troscopy are the leading techniques [1].
The thermal pasteurization of liquid (e.g.,
milk, water) is well established and satisfactory
for terminal decontamination/disinfection of such
commodities but inappropriate for solid foods,
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_12, © Springer India 2012
dry ingredients, and fresh foods known for raw
characteristics. The radiation pasteurization with
low doses of gamma (g) rays, X-rays, and electron
beam effectively controls food-borne pathogens.
Irradiation leads to the destruction of pathogenic
nonspore-forming food-borne bacteria and para-
sitic organisms, such as trichina, and protects the
consumers from microorganism-related diseases.
The application is based on the fact that ionizing
radiation causes very effective disruption of DNA
molecules in the nuclei of cells, rendering them
inactivate [2]. Irradiation has been examined
thoroughly by joint committees of the World
Health Organization (WHO), the United Nations
Food and Agriculture Organization (FAO), the
European Community Scientific Committee for
Food, Food and Drug Administration (USA), and
by House of Lords Committee. Food absorbs
energy when it is exposed to ionizing radiation;
the amount of energy absorbed is called “absorbed
dose,” which is measured in units called grays
(Gy), kilograys (kGy) (1 kGy = 1,000 Gy). The
energy absorbed by the food causes the formation
of short-lived molecules known as free radicals,
which kill microorganisms and also interact with
other food molecules. Thus, irradiation leads to
check microorganisms, insect gametes, and plant
meristems from reproduction and have preserva-
tive effects as a function of the absorbed radiation
dose (Table 12.1), while chemical- or other radi-
ation-induced changes are minimal [4].
An important reason for the relatively high
sensitivity of DNA to the ionizing radiation is
that DNA molecules are much larger than other
213
214 12 Radiation Detection in Herbals

Table 12.1 Doses for various applications of irradiation [3]

S. No. Preservative effects and types of application Dose (kGy)
1 Killing and sterilizing insects (disinfestations of food) 0.2–0.8
2 Prevention of reproduction of food-borne parasites 0.1–3.0
3 Decrease of after-ripening and delaying senescence of some fruit and vegetables; extension 0.5–5.0
of shelf life of food by reduction of microbial populations
4 Elimination of viable nonspore-forming pathogenic microorganisms (other than viruses) 1.0–7.0
in fresh and frozen food
5 Reduction or elimination of microbial population in dry food ingredients 3.0–10.0

molecular structures inside the cell. The damage
is either direct, caused by reactive oxygen from
hydroxyl radicals (OH−) originating from the
radiolysis of water, or indirect. In the case of
an indirect hit, the damage to the nucleic acids
occurs when radiation ionizes an adjacent mole-
cule, which in turn reacts with the genetic material.
In view of the fact that water is a major compo-
nent of most foods and microbes, it is often the
adjacent molecule that ends up producing a lethal
product. The ionizing radiation causes water mol-
ecule to lose an electron producing H2O+, which
immediately reacts with other water molecules to
produce a number of compounds, including hydro-
gen and hydroxyl radicals, molecular hydrogen,
oxygen, and hydrogen peroxide. Hydroxyl radi-
cals are very reactive and are known to interfere
with the bonds between nucleic acids within a
single strand or between opposite strands.
Although biological systems have a capacity to
repair both single-stranded and double-stranded
breaks of the DNA backbone, the damage occur-
ring from ionizing radiation is random and exten-
sive [5]. Therefore, recovery processes in bacteria
after their radiation damage are unlikely to occur.
The differences in sensitivity to radiation among
microorganisms are related to the differences in
their chemical and physical structure and in their
ability to recover from the radiation injury. The
amount of radiation energy required to control
microorganisms in food and food supplements,
therefore, varies depending on the resistance of
the particular species and the number of organisms
present. Besides inherent abilities of microorgan-
isms, several environmental factors such as the
composition of the medium, moisture content,
temperature during irradiation, and presence or
absence of oxygen significantly influence their
radiation resistance, particularly in vegetative
cells. The actual dose employed is a balance
between that what is needed and that what can
be tolerated by the product without objectionable
changes (e.g., off-flavors, texture changes, and
flavor alterations).
Microorganisms in Herbals
In stored herbals, microorganisms develop favor-
able conditions for themselves; such incidents
are more common with poorly stored products.
From a hygienic viewpoint such contaminated
material should be destroyed irrespective if active
principles have been affected or not. A summarized
discussion on the problem is as below; [6]
Spores of bacteria and molds are always
present in the air, and dried herbs are particularly
liable to be contaminated. Under satisfactory
storage conditions, their presence is not a problem;
however, for some crude drugs, certain pathogenic
bacteria are specifically limited or excluded, for
example, Escherichia coli in digitalis, tragacanth,
and gelatin and Salmonellae in digitalis as well as
cochineal. Certain species of molds, for example,
Aspergillus flavus and Fusarium tricinctum, pro-
duce extremely potent mycotoxins, which in the
past have been the cause of many deaths through
the consumption of overwintered moldy grain in
certain countries. The spores of such species have
been shown not to be common in vegetable drugs
and would be harmless anyway unless allowed to
develop through poor storage. With the exception
of chromogenic species of bacteria, for example,
Bacillus prodigiosus, which produces red patches
Microorganisms in Herbals
in starchy materials, the presence of actively
growing bacteria is not readily visible. However,
bacterial growth is usually accompanied on the
crude drug by growth of molds whose presence is
soon evident by the characteristic smell and by
the mass of clinging particles entrapped in the
mycelial hyphae. In most cases, in order to iden-
tify a particular mold which is proliferating in a
stored product, it is necessary to culture it on a
suitable medium, to obtain fruiting bodies for
examination. However, if the drug to be exam-
ined is infested to an advanced degree, then it
may be possible to make microscopic prepara-
tions directly from the sample. The principal
molds encountered with poorly stored drugs
include the genera Mucor (e.g., the gray mold, M.
mucedo), Rhizopus (e.g., black mold, R. nigri-
cans), Penicillium (e.g., blue mold, P. glaucum),
Aspergillus (e.g., green mold), and Saccharomyces
(yeasts). The spores of Mucor and Rhizopus can
be clearly seen on their stalks with a hand lens,
and the members of this group can usually be
immediately recognized because of the rapidly
growing light-colored, loose-textured colonies
which are usually white or light gray in color.
Aspergillus forms a mop-like head of spores, while
Penicillium produces a flat brush-like head.
The arachnids (mites) are found in stored
drugs usually in countless numbers, on examina-
tion with a hand lens. They can be examined
microscopically by clearing a sample of the powder
containing them with chloral hydrate reagent; the
outline and appendages remain distinct, and the
females can be seen to contain numerous larvae.
A commonly found members of the group are the
flour mite (Tyroglyphus farinae, Acarus siro),
which thrives on starchy and cereal products,
cheese mite (Tyroglyphus dimidiatus), and
smaller mites (Cheyletus eruditus) that are often
found with herbals and/products.
A number of small beetles are a common
source of infestation of stored herbals. Beetles
undergo a complete metamorphosis, from egg to
larva to imago, and are harmful for stored herbals
at both larval and adult stages. The most common
beetle found in many drugs, including gentian,
liquorice, and rhubarb, as well as leafy parts and
seeds, is Stegobium paniceum, the biscuit beetle
215
(name being derived from its frequent occurrence
in the biscuits of the old-time sailors). The Lasio-
derma serricorne is found in stored products
including ginger and liquorice.
A few species of the Lepidoptera attack stored
products, and although damage at the larval stage
only, the infestation can spread rapidly due to the
mobility of the adults. Some species, for example,
Ephestia kuehniella, flour moth, produce larvae
which, when fully grown, remain in the food mate-
rial where they overwinter and pupate. On the other
hand, the grubs of E. elutella, the cocoa moth, leave
the original food containers and during this process
they cover themselves with a web of silky threads.
They enter cracks and crevices where they spin
cocoons and finally emerge as adults.
A general practice to use cool–dry condition
for the storage of herbals and products is not
always safe, for example, the eggs of the flour
mite can survive several months at a temperature
of 0°C and 12 days at −10°C. Irradiation of herb-
als and products is currently in practice since two
decades. These radiations are limited to high-
energy photons (gamma rays of radionuclides
60
Co and, to a much smaller extent, 137Cs or X-rays
from machine sources with energies up to
5 MeV or accelerated electrons with energies up
to 10 MeV produced by electron-accelerating
machines), to produce the desired food preserva-
tive effects, without inducing radioactivity in
foods, food supplements, or packaging materials,
for commercial purpose. The radiation treatment
causes only minimal temperature rise in the
product and can be applied through packaging
materials including those that cannot withstand
heat, meaning that the radiation treatment can be
performed also after packaging; thus, recontami-
nation or reinfestation of the product is avoided.
Long-term animal feeding studies have demon-
strated that radiation-pasteurized/sterilized foods/
food supplements are safe and nutritious also for
humans, irradiated at doses below 10 kGy. The
Directive 1999/3/EC established a community
list of foods and food ingredients that may be
treated with ionizing radiation (EC 1999).
According to this directive, maximum allowed
overall average absorbed dose is 10 kGy for dried
aromatic herbs, spices, and vegetable seasonings.
216
The Food and Drug Administration (FDA), USA,
sets a limit for irradiation treatment of culinary
herbs, seeds, spices, vegetable seasonings, and
blends of these aromatic vegetable substances
that must not exceed 30 kGy. The radiation
pasteurization process for many foods has been
approved or endorsed by many world agencies
and associations such as the FDA, WHO, the
Codex Alimentarius Commission, the American
Medical Association, the Institute of Food
Technologies, and the health authorities in
approximately 40 countries [7].
Safety and Effectiveness of Irradiation
A mild treatment as a radiation dose of 1 kGy
represents the absorption of only enough energy
to increase the temperature of the product by
0.36°C. In fact, heating, drying, and cooking may
cause higher nutritional losses. Moreover, certain
carcinogenic aromatic and heterocyclic ring com-
pounds produced during thermal processing of
food at high temperatures were not identified in
food after irradiation. In general, food macronu-
trients (carbohydrates, proteins, and lipids) and
most micronutrients (mainly water-soluble and
fat-soluble vitamins) are not appreciably affected
by 10-kGy range ionizing dose with regard to
their nutrient contents. However, with higher
radiation doses (above 10 kGy, the exceeding
permitted limit in the EU), the structural proper-
ties of the fibrous carbohydrates can be degraded,
and lipids can become somewhat rancid, leading
to a loss of food quality [7, 8]. Moreover, the irra-
diation of lipids at high doses, and especially in
the presence of oxygen, can lead to the formation
of liquid hydroperoxides. The oxidation products
formed often have undesirable odors and flavors
(rancidity). It is known that the unsaturated fatty
acids are more prone to develop rancidity. Lipid
oxidation can be significantly reduced by freezing
and/by oxygen removal prior to irradiation. Among
micronutrients, thiamine is a subject of concern
as it is relatively sensitive to the effects of radia-
tion. Foods that contain thiamine (e.g., pork) are
suitable as indicators of food safety regarding the
irradiation treatment [7, 8], but minerals remain
12 Radiation Detection in Herbals
stable. Besides the nutritional and sensory values,
the wholesomeness (lack of mutagenicity, terato-
genicity, and toxicity) of irradiated foods has
been studied extensively. Neither short nor multi-
generational feeding studies have produced any
evidence of toxicological effects in mammals due
to ingestion of irradiated foods. In fact, multigen-
erational studies with animals have demonstrated
that the ingestion of irradiated foods is completely
safe and that the nutritive value remains essen-
tially unchanged. The data support the conclusion
that properly processed irradiated foods are whole-
some. Radiolytic changes in foods are minimal
and are predictable from the radiation chemistry
of principal food components. Furthermore,
possible radiolytic products derived, for exam-
ple, from lipids (most of which are saturated
and unsaturated hydrocarbons, aldehydes, and
2-alkylcyclobutanones) are neither unique nor
toxicologically significant in the quantities found
in irradiated foods. Irradiation is necessary for
the safety from microflora, and other benefits are
an increased shelf life (of fruits, vegetables, spices,
and meat), and a suitable alternative to chemical
treatments, especially for the decontamination of
fruits, vegetables, and spices [7].
Irradiation of Spices and Dried
Vegetable Seasonings
The aromatic oil-bearing plant parts, especially
essential oil-rich moieties, are known as spices
often collected from the developing countries
where harvest and storage conditions are poor
with respect to food hygiene. A very small amount
of these spices may be a potential source of
microbial pollution for food stuffs to which they
were added. Most spices are dried in the open
air and can become seriously contaminated by
air- and soil-borne bacteria, fungi, and insects.
Microorganisms of public health significance
such as Salmonella, Escherichia coli, Clostridium
perfringens, Bacillus cereus, and toxigenic molds
can also be present. Bacterial plate counts of
1–100 million per gram of spice are usual. Good
manufacturing practices during harvest and pro-
cessing could improve their hygienic quality, but
Irradiation Dose for Essential Oils and Organoleptic Characters
frequently not to an extent sufficient to obtain
an acceptable microbiological purity level.
Contaminated, dry plant ingredients cause seri-
ous troubles in the food-based industry. Therefore,
many commercial food processors fumigate
spices with methyl bromide to eliminate insects
or with ethylene oxide to eliminate bacteria and
molds. However, it has been found that both
methyl bromide and ethylene oxide are extremely
toxic compounds. Moreover, methyl bromide is
potentially capable of depleting the atmospheric
ozone layer. Ethylene oxide has been banned in
Europe because of safety and environmental
concerns, and its use for the treatment of ground
spice has been banned in the United States. The
US Clean Air Act and the Montreal Protocol of
the Vienna Convention require that any substance
listed as ozone depleting be withdrawn from
production and use by the year 2001. Spices,
herbs, and dried vegetable seasonings are cur-
rently treated with ionizing radiation to eliminate
microbial contamination. It was unambiguously
confirmed that the treatment with ionizing energy
is more effective against bacteria than the thermal
treatment and does not leave chemical residues
in the food product. Thus, ethylene oxide and
methyl bromide treatments can be effectively
replaced by food irradiation, which in fact is less
harmful to the spices than heat sterilization, as it
leads to the loss of thermolabile aromatic vola-
tiles and/causes additional thermally induced
changes (e.g., thermal decomposition or produc-
tion of thermally induced radicals) [7].
Irradiation Dose for Essential Oils
and Organoleptic Characters
Gas chromatography with flame ionization detec-
tion (GC/FID), gas chromatography–mass spec-
trometry (GC/MS), sensory evaluation, and
GC–olfactometry are in use to compare the impact
of irradiation on essential oils. In a case study, pow-
dered black pepper was irradiated with different
recommended doses of gamma rays (5 and 10 kGy,
respectively) and treated with microwaves for dif-
ferent periods (20, 40, and 75 s). The results obtained
indicate that irradiation treatment with controlled
217
doses of gamma irradiation is a safe and suitable
technique for decontamination of black pepper.
In comparison with microwave treatment, irradia-
tion does not result in extensive loss of flavor
compounds. In contrast to the radiation treatment,
the thermal treatment of black pepper (using 130°C
hot dry steam for 4 min, internal temperature of the
treated berries was 95°C) caused a significant
increase in the content of monoterpenes [7, 9].
These changes might have resulted from the forma-
tion of thermal isomerization products of some
terpenes. The qualitative composition of volatile
oils obtained from control, thermally treated sam-
ple, and from irradiated samples of black pepper at
various doses (up to 30 kGy) was identical. Statistical
analysis of the effects of irradiation on the total
content of volatile oils in spices showed no
significant differences between irradiated and non-
irradiated samples. As per Sadecka et al., the most
important changes were observed in the black pep-
per sample irradiated up to the dose of 30 kGy,
which resulted in a threefold increase of caryophyl-
lene oxide concentration and a parallel decrease of
sesquiterpene caryophyllene, in comparison with
an untreated sample [7, 10]. Nevertheless, such a
dose of ionizing energy exceeds three times the
level permitted by the EU legislation (EC 1999).
The observed changes induced in terpenes by irra-
diation could be explained by oxidation or hydroxy-
lation of aromatic rings in terpene molecules. The
products with low moisture content (10%) are prone
to the formation of free radicals by irradiation which
leads to an increased production of oxides and alco-
hols. In addition, the configuration of the side groups
on the terpene skeleton, especially the position of
double bonds and functional groups, can result in a
variety of compounds produced. The research has
demonstrated that gamma irradiation at the dose of
10 kGy (toxicologically and nutritionally confirmed
maximum safe dose) can eliminate microbial load
of spices without causing any significant organolep-
tic or chemical alterations [7].
Studies on dried basil leaf for essential oils by
Antonelli et al. observed that the composition of
essential oils treated at doses of 5 and 10 kGy
was different in comparison to the controlled
sample. They concluded that radiation caused
more evident changes in the composition profiles
218
than the microwave treatment. Subsequently, a
sensory test confirmed significant differences
between the extracts. The panelists preferred the
gamma-treated sample, while the microwaved
sample was least appreciated [7].
Radiation Quantity and Antioxidant
Activity
Spices (anise, cinnamon, ginger, liquorice, mint,
nutmeg, cumin, vanilla, etc.) are well-known
source of antioxidants used in daily life. In a case
study with the nonirradiated samples, the water
extracts of the irradiated spices at 1, 3, 5, and
10 kGy did not show any significant difference in
the antioxidant activity in the radical-scavenging
assay. The antioxidant properties of anise, cara-
way, cumin, and fennel essential oils extracted
from untreated, gamma-irradiated, and micro-
waved seeds was studied. Gamma-irradiation at
10 kGy and microwave treatments did not affect
the antioxidant property of the essential oils
under study. In addition, essential oils extracted
from gamma-irradiated fruits were more effective
antioxidants in sunflower oil than those produced
from microwaved fruits. The capsaicinoid content
in sun-dried and dehydrated paprika samples
that were irradiated at doses from 2.5 to 10 kGy
capsaicin, dihydrocapsaicin, and homodihydro-
capsaicin was increased by about 10% in the
samples irradiated at the dose of 10 kGy [7, 11].
The effects of irradiation by electron beam on
the color and the contents of volatile oils in five-
spice powder (prickly ash, star aniseed, cinna-
mon, clove, and fennel) and chili were assessed
by Lianzhong et al. [12]. Irradiation enhanced
the UV absorption of aqueous extracts of spices,
but the darkening phenomenon of spices due
to irradiation was temporary. In studies for the effect
of gamma irradiation at 10 kGy on the free
radical formation and the antioxidant contents
of aromatic herbs and spices (basil, bird pepper,
black pepper, cinnamon, nutmeg, oregano, pars-
ley, rosemary, and sage), Calucci et al., observed
that in a general increase of quinine radical
content [measured using electron paramagnetic
resonance (EPR) spectroscopy] in all samples
12 Radiation Detection in Herbals
investigated, and in a significant decrease of
the total ascorbate and carotenoids content of
some spices [13]. Results from different studies
have found no significant differences between
EPR spectra of the samples of white pepper,
sweet paprika, and nutmeg irradiated with elec-
tron beams or X-rays at 0–10 kGy irradiation.
Several studies using EPR spectroscopy to inves-
tigate free radicals formed by the gamma-
radiation treatment of ground black pepper,
oregano, all spice, ginger, and clove and to evalu-
ate the influence of the absorbed dose of gamma
radiation on the radical-scavenging potential of
alcoholic extracts, it was observed that radical-
scavenging (antioxidant) activity of spice extracts
was only slightly influenced by the gamma-
radiation treatment [7, 14].
Detection of Irradiated Foods
and Food Supplements
For the international trade of foods and food
supplements, there is a look for simple and reli-
able method to identify irradiated food stuffs.
Numerous studies have dealt with the detection
methods applicable to irradiated herbs and spices
and have also concluded that food irradiation as a
most acceptable radiologically, microbiologically,
and toxicologically safe technology. Nevertheless,
questions focusing on nutrient loss, free radical
and radiolytic by-product formation, and changes
of antioxidant properties during irradiation are
still being discussed in the scientific community.
In 1993, the European Commission gave a man-
date to the European Committee for Standrdization
(CEN) to standardize the methods for the detec-
tion of irradiated foods. These European standards
have been adopted by the Codex Alimentarius
Commission as general methods and are referred
to in the Codex General Standard for Irradiated
Foods in section 6.4 on “Post-irradiation verifi-
cation” (Code of Federal Regulation 2004) [7].
In case of spices, to measure the degree of
irradiation, the most important methods are vis-
cosity measurement, EPR/ESR, and TL. In rela-
tion to the formation of paramagnetic species
due to gamma irradiation, EPR/ESR spectroscopy
Detection of Irradiated Foodsand Food Supplements
represents a unique detection technique for
detection and characterization. In 2000, CEN
issued the EN 1787 EPR method for the detec-
tion of irradiated foods containing cellulose. The
gamma-radiation treatment of plant products
containing cellulose leads to the generation of a
typical three-line EPR signal, attributable to
cellulosic radicals. Later, more standardized
methods were published for the detection of
irradiated foods (i.e., EN 13708, EN 13751, and
EN 13784). EN 1784 and EN 1785 describe
methods for the detection of irradiated food con-
taining fat, and EN 1786 is a method for the
detection of irradiated food containing bone.
Unfortunately, their application is limited by the
lifetime of the radiolytically produced free radi-
cals [14]. Raffi and Stocker observed that, even
though electron spin resonance (RSR) is known
to be a very sensitive method, in the case of
spices, it did not lead to favorable results because
the main radio-induced signal decreased too fast
with the storage time and disappeared before the
maximal usual commercial storage time [15].
Polonia et al. showed a prolonged appearance of
cellulose peaks in dried paprika [16]. This enabled
to include the EPR method in the European
protocol. For the purpose of post-radiation
identification of cellulose-containing foods, the
presence of relatively weak satellite lines of this
“cellulosic” radical species was accepted as an
unambiguous evidence for gamma-radiation treat-
ment. However, it was observed that the EPR
intensity of the cellulosic triplet signal gradually
decreased with the storage time and that the rate
of disappearance was dependent on temperature,
humidity, the presence of oxygen, and other factors.
In general, the cellulosic EPR signal disappeared
within 70–90 days after the irradiation process.
This method was deemed to fail in the verification
of the gamma-radiation treatment, and, conse-
quently, it was recommended that TL techniques
should be used. Yordanov et al. pointed to the
different thermal behavior of EPR signals of
nonirradiated and gamma-radiation-treated foods
containing cellulose, even after a long storage
period after radiation, when the specific cellulosic
EPR signal is extremely low and recommended
219
this technique as a method to identify gamma-
radiation-processed foods [17].
The utilization of EPR spectroscopy and vis-
cometry (with two different sample preparation
methods) to detect irradiated black pepper
samples concluded that the identification of irra-
diation at doses above 8 kGy is possible using
these methods, at least within 1 month of post-
irradiation storage at ambient temperature.
The viscosity measurement was reported to be a
promising method for the identification of irradi-
ated spices [7] and described a method using
heat gelatinization of starch in different spices.
In agreement with their findings, Farkas et al.
reported a dramatic decrease in the dispersion
viscosity of heat-gelatinized suspensions of
several irradiated spices with the starch content
compared to that of unirradiated samples. This
approach may provide a relatively simple diag-
nostic technique for the detection of irradiation
treatment of starch-containing spices. Because
the effect mentioned seems to be related to the
radiodepolymerization of starch in the irradiated
spices, additional analytical techniques have
been tested to investigate the starch damage in
black and white peppers [7]. The colorimetrically
determined reducing sugar content as well as
the alcohol-induced turbidity of hot-water extracts
indicated increased starch damage in the pepper
samples as a function of the irradiation dose.
However, the effect of irradiation had a less
dramatic response in these tests than in the
viscometric test. The moisture content influences
partial radiodepolymerization of starch. According
to the experimental results, the technique of dif-
ferential scanning calorimetry (DSC), measuring
the energy and temperature characteristics of heat
gelatinization of starches, cannot rival the sensi-
tivity of viscometric measurements in the detec-
tion of radiation-induced changes, suggested an
alternative method to that of heat gelatinization
of starch, which is less time-consuming because
it does not require heat gelatinization. In the case
of cinnamon and allspice (15% suspensions,
particle size less than 0.16 mm), the apparent vis-
cosities seemed to be as sensitive to the irradiation
dose as those of heat-gelatinized spices [7].
220
Fig. 12.1 Energy-level presentation of TL process, showing the thermally activated luminescence. N = concentration of
electron traps with energy Ee, M = concentration of hole traps with energy Ep
A comparison of three physical methods
[viscometry, DSC, NIR (near-infrared spectro-
photometry)] used in the identification of irradi-
ated spices (cinnamon and allspice) revealed that
the apparent viscosity test demonstrated the best
response sensitivity when applied to samples
irradiated with different doses. The storage time
did not influence the apparent viscosity values.
The identification limit of the viscometric method
was determined at 2–3 kGy, whereas the limit of
NIR spectrophotometric method was determined
at 4–5 kGy, respectively. These two methods
enabled to distinguish and correctly order the
irradiated samples. Thermal behavior of free
organic radicals induced in irradiated black pepper
was studied and observed that the radical evolution
in the irradiated pepper obeys a single exponential
function and yields a unique time constant. In a
study, the use of TL, EPR spectroscopy, and vis-
cosimetric measurements to determine whether
or not a spice had been irradiated confirmed that
TL, using the EN 1788 (2001) official protocol,
with an alternative method for the extraction of
mineral impurities, led to the proof of irradiation.
EPR can be used as a proof of irradiation just up
to a few weeks after irradiation and useful only
for some spices [7].
In view of the increasing use of irradiation
technology for preservation of herbs and prod-
ucts, the availability of analytical techniques for
identifying irradiated or nonirradiated moiety is
of much importance for both the regulatory
12 Radiation Detection in Herbals
authorities and consumers. Several detection
methods, broadly categorized as chemical (com-
parative antioxidant properties with reference
standard), physical (e.g., viscosity measurement),
and biological (e.g., antimicrobial against the
reference standard), have been developed for
the identification of irradiated foods. Among the
physical techniques PPSL, TL measurements,
and EPR/ESR spectroscopy are in practice.
Thermoluminescence
Thermoluminescence (TL) is the thermally stim-
ulated emission of light from an insulator or a
semiconductor following the previous absorption
of energy from ionizing radiation. The TL process
can be understood in terms of the band structure
model of insulators. In a pure insulator, there are
two relevant energy bands: (1) an almost com-
pletely filled valence band and (2) an almost
empty conduction band. The two energy bands
are separated by a forbidden gap, which means
that between these two bands there are no elec-
tronic energy levels. Transitions of electrons
between the valence band and the conduction
band are allowed, and they produce free electrons
in the conduction band and free holes in the
valence band (Fig. 12.1).
A large number of minerals (e.g., quartz, feld-
spar, calcite, flint) emitting light energy upon warm-
ing is known as thermoluminescence. The TL of
Detection of Irradiated Foodsand Food Supplements
minerals is roughly proportional to the irradiation
dose to which they had been exposed. The TL
method for the detection of irradiated herbs and
herbal products is based on the isolation of min-
erals (mineral debris) from the herb/herbal prod-
ucts and estimate the energy absorbed due to
irradiation, if any, as minerals store energy dur-
ing irradiation by trapping charge carriers in an
excited state. On controlled heating of these
minerals, after isolation, charge carriers are stim-
ulated to return to the ground state, thereby emit-
ting some of the energy as light. This light is
detected by a sensitive photon counter, and light
emission is recorded, known as glow curve. The
first glow curve (TL1) is compared with a second
glow curve (TL2) obtained by a second TL
measurement of the isolated minerals following
their exposure to a defined radiation dose. This
normalization procedure allows for differences in
mineral composition, for example, varying
amounts of feldspar or quartz and/in weight of
the aliquots of isolated mineral grains. A com-
parison of size and shape of the two glow curves
reveals whether the sample from which the min-
eral grains were isolated has been irradiated or
not (Figs. 12.2, 12.3). The TL glow ratio which is
the ratio of integrated TL intensities of glow 1(TL
1) to glow 2 (TL2), evaluated over a defined tem-
perature interval, is typically greater than 0.5 for
irradiated and generally below 0.1 for nonirradi-
ated samples [18]. TL measurement is a time-
consuming and cumbersome method. TL proved
to be the most reliable method as it was success-
fully used to distinguish all kinds of irradiated
samples from the nonirradiated control samples.
Pulse Photo-Stimulated Luminescence
In pulse photo-stimulated luminescence (PPSL),
the release of trapped energy from minerals
exposed to ionizing radiation is optically stimu-
lated by electromagnetic radiation of appropriate
wavelength, and the emission of stored energy is
recorded by a sensitive detector. The theoretical
principle is analogous to the two-stage process of
photon emission from infrared-stimulated feld-
spar where the photo excitation of charge from
221
the ground state of an electron trap to an excited
state is followed by elevation of the electron to
the conduction band by phonon absorption. PPSL
analysis takes just a few minutes and hence allows
for multiple measurements to be performed.
PPSL detection method has been used for rapid
screening of many irradiated foods including
brown shrimps, herbs, spices, seasonings, and
shellfish. In a study, 90% of 45 nonirradiated and
irradiated spices and seasonings were successfully
identified based on simple PPSL measurement.
Irradiated white ginseng powder, pepper powder,
potato, soybean, dried fig, and chestnut were also
successfully distinguished from nonirradiated
samples using PPSL [19].
Electron Paramagnetic Spin Resonance
Spectroscopy
Electron paramagnetic spin resonance (EPR/
ESR) spectroscopy technique is widely used for
identification of foodstuffs, for free radicals, and
molecules with an odd (unpaired) number of elec-
trons. Such moieties can be produced by high-
energy radiation and are detectable by EPR/ESR
method. There is a relation among free radicals
and quality of life and health. But some foodstuffs
have paramagnetic properties naturally, and they
show a single EPR spectral signal before irradia-
tion. Antioxidants are molecules which can safely
interact with free radicals and terminate the chain
reaction before vital molecules are damaged.
Antioxidants may diminish the energy of the free
radical, stop the free radical from reacting at the
potential side, and minimize the damage caused
by free radicals.
In a case study to understand the EPR spectral
properties of nonirradiated and gamma-irradiated
dry plants, cress seeds and mistletoe have been
studied, and the differences between nonirradiated
and irradiated samples were determined [19].
It has been observed that EPR spectra of these
nonirradiated samples have very weak signal, but
these plants have an intense EPR resonance line
after irradiation. Mistletoe and cress seeds were
selected for studies, and each of the samples was
taken in polyethylene bags. These samples were
222
Fig. 12.2 (a, b) Typical TL (glow 1) curves of silicate
minerals isolated from mushrooms: 1 champignon, 2 por-
cini or yellow boletus, 3 chanterelle, 4 black morel, 5
Suillus or slippery Jack, 6 bay boletus, 7 Chinese black
prepared also for irradiation. Irradiation was
performed in air and at room temperature with a
60
Co gamma-ray source. For both of the samples,
irradiation dose rate was from 0.5 to 2.5 kGy.
All EPR measurements were performed at room
temperature on Varian X-Band EPR spectrometer
operating at a microwave frequency of 9.53 GHz,
with 100-kHz field modulation, using a standard
rectangular cavity. Samples were inserted in
quartz tubes (3–4 mm), and reference material
containing Mn2+ was fixed in EPR cavity; this is
due to the fact that position of samples in cavity
12 Radiation Detection in Herbals
mushroom (mun), 8 Chinese morel (cloud ear), 9
Craterellus (black trumpet) [18], where (a) nonirradiated
samples and (b) samples irradiated with 7 kGy of 60Co
gamma rays
affects the intensity of lines. Samples of mistletoe
and cress seeds have a weak EPR signal with
g = 2.0046 ± 0.0005 value before irradiation. Sugar-
like EPR spectrum was not observed after irradi-
ation. Both of the samples have a resonance line
in EPR spectra after irradiation appeared. The
only difference from nonirradiated properties is
intensity (Fig. 12.4).
European Committee has adopted the six pro-
tocols; three of these (CEN, 1997, 2000, 2001a)
use in EPR spectroscopy for irradiation studies,
the first one for bone, the second for cellulose
Detection of Irradiated Foodsand Food Supplements 223

Fig. 12.3 (a) TL glow curves of minerals separated from erals separated from fruit, ramulus, folium, flower, spike,
the irradiated (0–50 kGy) root, rhizome, and bark of ten peel, and whole plant of ten different dry medicinal herbs
different dry medicinal herbs. (b) TL glow curves of min- irradiated at different doses (0–50 kGy) [1]

Fig. 12.4 EPR spectra

of mistletoe (a) nonirradiated
(b) irradiated [19]
224
Scrophularia root
50 kGy
25 kGy
10 kGy
5 kGy
0 kGy
5mT
Artemisiae argyi folium
50 kGy
25 kGy
10 kGy
5 kGy
0 kGy
5mT
Fig. 12.5 ESR spectra of nonirradiated and irradiated
Scrophularia root, Scutellaria root, Artemisiae argyi
folium, and Alisma rhizome. Only one intense singlet
containing foodstuffs, and the third for foodstuffs
containing crystalline sugar. In all protocols only
X-band EPR technique is used. Following these
protocols, the problem that must be solved by
EPR is on a qualitative level, whether an appro-
priate food sample has been irradiated before or
not. For an ideal case, nonirradiated samples con-
taining hard tissues were selected for the herbals
and herbal products, having EPR signal that is
stable. Identification procedure of dry spices and
some plants is based on the fact that, whereas
nonirradiated samples exhibit only one weak
singlet EPR signal, its amplitude significantly
increases after irradiation together with the
appearance of two weak satellite lines. These lines,
originating from a triplet spectrum whose central
line is overlapped on the initial line, are attributed
12 Radiation Detection in Herbals
Scutellaria root
50 kGy
25 kGy
10 kGy
5 kGy
0 kGy
5mT
Alisma rhizome
50 kGy
25 kGy
10 kGy
5 kGy
0 kGy
5mT
line can be seen in ESR spectra of the irradiated
Scrophularia root, Scutellaria root, and Artemisiae argyi
folium [1]
to free radicals of cellulose generated by irradiation.
The obtained triplet EPR spectrum is characterized
with g = 2.006070.0005 and a = 3,070.5 G. Just
the presence of these two satellite lines is consid-
ered in the protocol (CEN, 2000) as unambigu-
ous evidence for previous radiation treatment of
plants [19].
It is imperative to consider the carbohydrate
spectra also for detection of irradiation status of
herbal products using EPR/ESR spectrometry.
ESR spectra of four irradiated samples of
Scrophularia root, Scutellaria root, Artemisiae
argyi folium, and Alisma rhizome did not show
any resemblance with any established EPR/ESR
signals, such as cellulosic and sugar- or carbohy-
drate-like EPR/ESR spectrum, typical for most
of the irradiated plant products [1] (Fig. 12.5).
Detection of Irradiated Foodsand Food Supplements 225

Table 12.2 Suitability of detection methods to identify irradiated dried mushrooms [18]
S. No. Sample TL EPR PPSL
1 Champignon (Agaricus boletus) + + −
2 Porcini (Boletus edulis) + ± +
3 Chanterelle (Cantharellus cibarius) + ± +
4 Black morel (Morchella conica) + − +
5 Suillus (Suillus luteus) + ± −
6 Bay boletus (Xerocomus badius) + ± ±
7 Chinese black mushroom (Lentinus edodes) + − −
8 Chinese morel (Auricularia polytricha) + − +
9 Craterellus (Craterellus cornucopioides) + + +

For irradiated Scrophularia root, Scutellaria
root, and Artemisiae argyi folium, the spectra
were characterized by one intense singlet at
g = 2.0045 ± 0.0007. The nonirradiated line due to
lignin also appeared as a singlet with the same
spectroscopic parameters. Thus, the irradiated
products of these three samples could not be
distinguished from the nonirradiated ones using
X-band ESR spectroscopy. As EPR/ESR spec-
trometry is not calibrated, no definite judgment
about the irradiation status can be made based on
change in ESR spectrum intensity [1].
The life time of the radiation-induced radicals
strongly depends on the storage conditions (as
hvumidity and temperature) before and after irra-
diation, and generally their EPR spectrum disap-
pear within 70–90 days after irradiation. According
to some reports, however, the above satellite EPR
lines are not observed or disappeared very shortly
after irradiation of the food samples. Therefore, the
presence of the triplet EPR spectrum of cellulose
free radicals if recorded is unambiguous evidence
that the sample under study has been irradiated, but
its absence cannot always be considered as the
opposite. In this case, following European protocol
TL should be used (CEN, 2001b) which is time
consuming. In order to extend the period of
identification by using EPR, the central EPR line is
useful as a proof for previous radiation treatment of
herbs and spices, since its intensity remains enor-
mously high even after the disappearance of the
satellite lines. In this approach, the difference in the
intensity of the singlet EPR line recorded before
and after heating of the sample up to 60°C is moni-
tored. In the cases when the results obtained by the
EPR spectrometry are not definitive with respect to
radiation treatment, the method of TL (CEN,
2001b) can be used. It is unambiguous but is very
slow and expensive, and a general recommendation
is to use first EPR and if results are not clear to do
TL (Table 12.2).
Food and food supplement irradiation has
been shown to be a safe and effective process
in controlling microbial contamination without
adverse side effects and chemical residues that
can be used to improve the safety of supply in the
markets. There is a need to standardize analytical
procedures for organoleptic quality of irradiated
foods, and food supplements mainly spices, in
the dependence on the dose used for ionizing
radiation. In this context, it is necessary to stan-
dardize analytical procedures for an unambiguous
identification of gamma-radiation treatment and
its influence on food quality, in order to obtain
results that are intercomparable with the aim of a
better protection and objective consumer informa-
tion, respecting their freedom of choice. Scientists
have to work more to develop a cheap and validated
method to determine the irradiation potential on
food supplements, for example, Cassia angustifolia
extract (BP, USP grade), Coleus forskohlii extract,
Garcinia cambogia extract (as calcium salt), and
Garcinia mangostana extract in market as food
supplement.
226
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